2.1 FUNCTIONAL DOMAINS IN PROTEINS
Many biological processes involve the specific binding of proteins to one another. Examples of such processes are signal transduction, transcription, DNA replication, cytoskeletal organization, membrane transport, etc. In many cases it has been shown that specific binding is mediated by small portions of the proteins involved and that these portions can function to a large extent independently of the rest of the proteins. Such independent portions of proteins, mediating specific recognition or binding of one protein by another, have come to be called "functional domains". A variety of functional domains have been characterized to a variety of levels of understanding. Some of these are described below.
Src homology 2 domains (SH2) domains are short (about 100 residues) amino acid sequences that were originally found in the non-membrane bound tyrosine kinase Src. Since then they have been shown to occur in about 20 other proteins. SH2 domains recognize certain phosphotyrosine-containing sites on proteins. Proteins containing SH2 domains participate in a variety of signalling pathways. For reviews discussing SH2 domains see Pawson, 1995, Nature 373:573-580; Cohen et al., 1995, Cell 80:237-248; Pawson and Gish, 1992, Cell 71:359-362; Koch et al., 1991, Science 252:668-674.
Src homology 3 (SH3) domains are another class of short amino acid sequences that were originally found by comparing the amino acid sequence of the Src protein with the sequences of Crk, Phospholipase C-.gamma., .alpha.-Spectrin, Myosin IB, Cdc25, and Fus1 (Lehto et al., 1988, Nature 334:388; Mayer et al., 1988, Nature 332:272-275; Stahl et al., 1988, Nature 332:269-272; Rodaway et al., 1989, Nature 342:624). In addition to Src, almost 30 proteins are known to contain SH3 domains and these proteins perform a wide range of functions.
For reviews discussing SH3 domains see Pawson, 1995, Nature 373:573-580; Cohen et al., 1995, Cell 80:237-248; Pawson and Gish, 1992, Cell 71:359-362; Koch et al., 1991, Science 252:668-674.
SH3 domains have been shown to specifically bind certain proline-rich amino acid sequences (Chen et al., 1993, J. Am. Chem. Soc. 115:12591-12592; Ren et al., 1993, Science 259:1157-1161; Feng et al., 1994, Science 266:1241-1247; Yu et al., 1994, Cell 76:933-945; Sparks et al., 1994, J. Biol. Chem. 269:23853-23856; Sparks et al., 1996, Proc. Natl. Acad. Sci. USA 93:1540-1544). However, in general, the homology between different sequences that bind SH3 domains tends to be low.
This low homology would explain the specificity that has usually been observed for the interactions between SH3 domains and their natural ligands. Generally, a sequence that is identified by screening for binders to a particular SH3 domain will bind to that particular SH3 domain much more strongly that it binds to other SH3 domains. For example, Cicchetti et al., 1992, Science 257:803-806 probed a .lambda.gt11 cDNA expression library with a glutathione S-transferase fusion protein containing the 55 amino acid SH3 region of Abl and isolated two clones that produced proteins capable of specifically binding the Abl SH3 domain. Analysis of one of the clones uncovered the region of the encoded protein responsible for binding to the SH3 domain. This region, as part of a glutathione S-transferase fusion protein, bound the SH3 domain from Abl very strongly, the SH3 domain from Src less well, and the SH3 domains from Crk and neural Src very weakly.
Pleckstrin is the major substrate for Protein Kinase C in platelets. Two domains of about 100 amino acids in Pleckstrin have been found to have counterparts in a number of signal transduction and cytoskeletal proteins. These domains are known as Pleckstrin homology, or PH, domains (Haslam et al., 1993, Nature 363:309-310; Mayer et al., 1993, Cell 73:629-630). Although the sequence homology between PH domains from various proteins is low, structural studies have shown that PH domains fold into a similar conformation containing two antiparallel .beta. sheets and a long C-terminal .alpha. helix (Gibson et al., 1994, Trends Biochem. Sci. 19:349-353). Among the proteins that have been found to have PH domains are a number of proteins with important roles in signal transduction or cytoskeletal architecture, e.g., Spectrin, Dynamin, Phospholipase C-.gamma., Btk, RasGAP, mSOS-1, Rac, Akt.
Leucine zippers consist of alpha helical regions of proteins in which a leucine residue appears at every seventh position along the helix. The leucines interdigitate with leucines from the leucine zipper of a different protein or another molecule of the same protein, leading to dimerization of the proteins containing the leucine zippers. Leucine zippers have been found in a number of proteins that are believed to function as transcription factors, e.g., C/EBP, Myc, Fos, Jun, GCN4. In many of these proteins, dimerization through leucine zippers has been shown to be necessary for the DNA binding activity of the transcription factor.
The binding of leucine zippers exhibits specificity in that some leucine zippers preferably bind to certain other leucine zippers. For example, the Jun-Fos heterodimer formed by the binding of the leucine zippers of Fos and Jun forms in reference to a Jun--Jun homodimer formed by the binding of the leucine zippers of two Jun proteins.
Fas/APO-1(CD95) is a member of a class of transmembrane receptors that have been shown to be involved in the phenomenon of programmed cell death or apoptosis (Itoh et al., 1991, Cell 66:233-243). The tumor necrosis factor receptor 1 (TNFR-1) is also a member of this class (Baglioni, C., 1992, "The Molecules and Their Emerging Roles in Medicine," in Tumor Necrosis Factors, B. Beutler, ed. (New York: Raven Press). Itoh, N. and Nagata, S., 1993, J. Biol. Chem. 268:10932-10937 have shown that certain amino acid sequences in the cytoplasmic domain of Fas/APO-1(CD95) are required for the programmed cell death response mediated by this receptor. Tartaglia et al., 1993, Cell 74:845-853 proposed that a similar region in TNFR-1 also is responsible for programmed cell death. This region of similarity between Fas/APO-1(CD95) and TNFR-1 has come to be called the cell death domain.
Three groups have used the yeast two-hybrid system to clone genes whose products specifically bind to the cell death domains of Fas/APO-1(CD95) and TNFR-1 (Hsu et al., 1995, Cell 81:495-504; Chinnaiyan, et al., 1995, Cell 81:505-512; Stanger et al., 1995, Cell 81:513-523). These genes were shown to induce apoptosis when overexpressed in certain cell types, a result which argues that they are intracellular transducers of death signals from Fas/APO-1(CD95) and TNFR-1.